1. Field of the Invention
The present invention relates to improved methods for detecting and treating tumors and lesions and obtaining biopsy material in the course of intraoperative, laparoscopic, intravascular, and endoscopic examination using a small detection instrument or monitor. In preferred embodiments, the methods utilize a labeled divalent single chain antibody fragment or subfragment with a molecular weight of 85,000 daltons or less that specifically binds to an antigen produced by or associated with the tumors or lesions. Alternatively, bispecific F(ab)2 and F(abxe2x80x2)2 fragments can also be used for pre-targeting according to the present invention, if non-targeted fragments are cleared, and a bivalent diagnostic hapten is then administered to facilitate target detection and possibly other procedures within 48 hours of the first injection.
2. Description of the Prior Art
Surgical resection remains the primary curative approach in the management of cancer. Radioimmunodetection (RAID) is used to locate and stage tumors, and to monitor post-operative patients, by external imaging, after injection of a radiolabeled antibody. Antibodies and/or antibody fragments which specifically bind antigens produced by or associated with tumors (xe2x80x9canti-cancer antibodiesxe2x80x9d) are used as carriers for radiolabels in RAID. It will be appreciated, that a tumor antigen can serve as a target for an antibody carrier even if it is not present in serum in a detectable amount.
Resolution is affected by several factors that can limit the size of a tumor, especially a metastasis, which can be imaged by RAID. Non-invasive RAID is inherently limited by the distance between the radiation detector and the tumor. In the case of small, deep-seated metastatic tumors, this becomes the limiting factor in their detection.
Second-look surgery has been practiced where recurrence of a previously excised primary tumor was indicated by elevated levels of tumor marker, e.g., carcinoembryonic antigen (CEA) . Recently, a small gamma detection probe has been developed which is capable of detecting gamma emission at short distances. Its intraoperative use in second-look surgery has been reported to provide important information to the surgeon for determining safe margins for resection and for detecting small metastases, by Aitken et al., Dis. Colon Rectum, 27, 279-282(1984).
Nevertheless, elevated background radiation levels can interfere with and vitiate the advantage of short measuring distances in this technique. In addition, non-specific immunoglobulin uptake by tumor tissue can complicate diagnosis.
U.S. Pat. No. 4,782,840 discloses a method for reducing the effect of elevated background radiation levels during surgery. The method is to inject the patient with antibodies specific for neoplastic tissue and which are labeled with radioisotopes having a suitably long half-life, such as Iodine-125. After injection of the radiolabled antibody, the surgery is delayed at least 7-10 days, preferably 14-21 days, to allow any unbound radiolabeled antibody to be cleared to a low bloodpool, background level.
U.S. Pat. No. 4,932,412 discloses methods for reducing or correcting for non-specific background radiation during intraoperative detection. The methods include the administration to a patient who has received a radiolabeled primary antibody, of a contrast agent, subtraction agent or second antibody which binds the primary antibody.
Tumors can be detected in body cavities by means of directly or indirectly viewing various structures to which light is delivered and then collected. Lesions at any body site can be viewed so long as nonionizing radiation can be delivered and recaptured from these structures.
The prior art discloses improvements of such imaging approaches by using certain dyes that are accreted by lesions, such as tumors, which are in turn activated by a specific frequency of light. These improvements are described in Dougherty et al., Cancer Res. 38:2628, 1978; Dougherty, T. J., Photochem. Photobiol. 45:879, 1987; Jori and Perria, eds., Photodynamic Therapy of Tumors and Other Diseases; Padua: Libreria Progetto, 1985; Profio, Proc. Soc. Photoopt. Instr. Eng. 907:150, 1988; Doiron and Gomer, eds., Porphyrin Localization and Treatment of Tumors; New York: Alan Liss, 1984; Hayata and Dougherty, Lasers and Hematoporphyrin Derivative in Cancer; Tokyo: Igaku-Shoin, 1984; and van den Bergh, Chem. Britain 22:430, 1986, incorporated herein in their entirety by reference.
These dyes are injected, for example, systemically, and laser-induced fluorescence can be used by endoscopes to detect sites of cancer which have accreted the light-activated dye. For example, this has been applied to fluorescence bronchoscopic disclosure of early lung tumors (Doiron et al., Chest 76:32, 1979, included herein by reference; and references cited above).
It is known that dyes can be attached to antibodies for a more specific binding to certain tissues and cells, including malignant and normal cells, depending upon the discriminatory power of the antibodies in question. In cancer, such labeled antibodies have been used in flow cytometry and in immunohistology to stain malignant cells with many different kinds of anticancer antibodies, as described, for example, in Goding, Monoclonal Antibodies: Principles and Practice; London/New York, Academic Press, 1983; Ferrone and Dierich, eds., Handbook of Monoclonal Antibodies; Park Ridge, N.J., Noyes Publications, 1985; Wick and Siegal, eds., Monoclonal Antibodies in Diagnostic Immunohistochemistry; New York/Basel, Marcel Dekker, 1988, incorporated herein in their entirety by reference. Fluorescent and other chromagens, or dyes, such as porphyrins sensitive to visible light, have been used to detect and even treat lesions by directing the suitable light to the tumor or lesion (cited above). In therapy, this has been termed xe2x80x9cphotoradiation, phototherapy, or photodynamic therapy (Jori and Perria, eds., Photodynamic Therapy of Tumors and Other Diseases, Padua: Libreria Progetto, 1985; van den Bergh, Chem. Britain 22:430, 1986).
Monoclonal antibodies have been coupled with photoactivated dyes for achieving a photodetection or photocopy (Mew et al., J. Immunol. 130:1473, 1983; idem., Cancer Res. 45:4380, 1985; Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744, 1986; idem., Photochem. Photobiol. 46:83, 1987; Hasan et al., Prog. Clin. Biol. Res. 288:471, 1989; Tatsuta et al., Lasers Surg. Med. 9:422, 1989; Pelegrin et al., Cancer 67:2529, 1991xe2x80x94all incorporated in their entirety herein by reference). However, these earlier studies did not include use of endoscopic imaging and/or therapy or biopsy applications, especially with the use of antibody fragments or subfragments.
Further, there is a need in the art to utilize antibodies and antibody fragments that provide superior targeting specificity and affinity but which are cleared quickly and naturally through the kidneys or which can be cleared quickly with clearing agents, so that targeting can be effected within 48 hours.
A need continues to exist for simple methods which permit enhanced resolution to be achieved for close range intraoperative, intravascular, and endoscopic lesion detection, biopsy and therapy.
A need also exists for improved methods for detection and therapy of tumors.
A need also exists for methods to enable a clinician to intraoperatively, laparoscopically, intravascularly or endoscopically detect and treat non-malignant pathological lesions.
A need further exists for methods to accurately locate lesions in a patient to guide the biopsy implement to the lesion during a biopsy procedure.
A principal object of the present invention is to provide methods for close-range intraoperative, laparoscopic intravascular, and endoscopic detection of tumors whereby discrimination between tumor and non-tumor tissue is enhanced so that smaller tumors can be detected and appropriate margins can be determined more accurately to permit resection, irradiation, biopsy and/or tumor removal during the procedure and within hours of an injection of labeled protein, whereby surgical, intravascular, laparoscopic, endoscopic evaluation is not delayed.
Another object of the present invention is to provide a method for intraoperative, laparoscopic, intravascular or endoscopic detection, biopsy and therapy of tumors whereby the procedure can be selectively conducted within hours of the injection.
Another object of the present invention is to provide methods for close-range intraoperative, laparoscopic, intravascular, or endoscopic detection and treatment of non-malignant pathological lesions.
Another object of the invention is to provide improved laser therapy, radioimmunotherapy, immunochemotherapy, and/or biopsy procedures for lesions.
Upon further study of the specification and appended claims, further objects and advantages of this invention will become apparent to those skilled in the art.
These objects can be attained in a method for close-range tumor detection, biopsy and treatment during an operative, laparoscopic, intravascular or endoscopic procedure. The method comprises injecting a patient subject to such a procedure parenterally with an effective amount of a labeled protein, such as an antibody fragment or subfragment, which specifically binds a substance produced by or associated with a targeted tumor. While the procedure is conducted, the accessed interior of the patient is scanned at close-range with a detection means for detecting the presence of the labeled antibody fragment or subfragment. The sites of accretion of the labeled antibody fragment or subfragment are located by detecting elevated levels of the label at such sites with the detection means.
These objects can be attained in a method for close-range intraoperative, laparoscopic, intravascular, and endoscopic tumor detection, wherein a patient subject to such a procedure is injected parenterally with an effective amount of a labeled antibody fragment which specifically binds an antigen produced by or associated with a tumor; the procedure is conducted, preferably, within 48 hours of the injection when unbound radiolabeled antibody fragment in the patient""s circulation is reduced at least 50%; the surgically exposed, laparoscopically, or intravascularly or endoscopically accessed interior of the patient is scanned at close range with a label detection means; and the sites of accretion of the labeled antibody fragments are located by detecting elevated levels of label at such sites with the detection means.
In another related aspect, the invention provides a method for close-range detection during an intravascular, operative, laparoscopic, or endoscopic procedure of a non-malignant pathological lesion, wherein a patient subject to the procedure is injected parenterally with an effective amount of an antibody fragment, subfragment or whole antibody, labeled with a labeling agent and which specifically binds an antigen produced by or associated with the lesion, the accessed interior of the patient is scanned at close-range with a detection means for detecting the presence of the labeling agent, and the sites of accretion of the labeled antibody fragments or labeled whole antibody are located by detecting elevated levels of the labeling agent at such sites with the detection means.
The invention also provides a kit suitable for use in an intraoperative, laparoscopic, intravascular, and endoscopic procedure. The kit comprises a first vial containing a sterile injectable preparation for human use comprising an antibody fragment capable of being conjugated with a labeling agent, and a second vial containing a sterile injectable preparation for human use comprising a whole antibody capable of being conjugated with a labeling agent different from the labeling agent of the first vial.
In a related aspect, the invention provides a kit suitable for use in an intraoperative, laparoscopic, intravascular, or endoscopic procedure. The kit comprises a first vial containing a sterile injectable preparation for human use comprising a labeled antibody fragment and a pharmaceutically acceptable carrier, and a second vial containing a sterile injectable preparation for human use comprising a whole antibody labeled with a labeling agent and a pharmaceutically acceptable carrier, wherein the labeling agent of the second vial differs from the labeling agent of the first vial.
In another related aspect, the invention provides an improved method of detection of lesions using endoscopic, laparoscopic, intravascular catheter or surgical procedures. The method comprises injecting a patient to undergo such a procedure with a protein, such as an antibody, antibody fragment or subfragment, labeled with a fluorescent agent or dye, wherein the protein accretes at the lesion; permitting the labeled protein to accrete; and detecting the label with a light source supplied via the endoscope, laparoscope, or intravascular catheter or during the surgical or during the biopsy procedure.
In another related aspect, the invention provides an improved method of detection and treatment of lesions using an endoscope, laparoscopic, or intravascular catheter. The method comprises injecting a patient to undergo such a procedure with an agent capable of detection which preferentially accretes at the lesion; permitting the agent to accrete at the lesion; detecting the agent with a detection means supplied via the endoscope, laparoscopic, or intravascular catheter; and treating the lesion by brachytherapy administered through the endoscope, laparoscopic, or catheter.
In another aspect, the invention provides an improved method of treatment of lesions using endoscopic, laparoscopic, intravascular catheter or surgical procedures. The method comprises injecting a patient to undergo such a procedure with an antibody or antibody fragment labeled with a photoactive agent, wherein the antibody or fragment preferentially accretes at targeted lesions, permitting the labeled antibody or fragment to accrete, and activating the photoactive agent with a light source supplied via the endoscope, laparoscopic, or intravascular catheter or during the surgical procedure.
In another aspect, the invention provides an antibody fragment coupled with a photoactive agent.
In another aspect, the invention provides an improved method of detection and treatment of lesions using endoscopic, laparoscopic, intravascular catheter or surgical procedures.
The method comprises injecting a patient to undergo such a procedure with a first composition comprising either a streptavidin- or avidin-conjugated antibody, biotinylated antibody to be used in conjunction with avidin and biotin, bifunctional antibody, antibody-hapten complexes, or enzyme-conjugated antibody, wherein the antibody is an antibody or antibody fragment which specifically binds a marker produced by or associated with the lesion; after the first composition accretes at the targeted lesion, injecting a second composition which bears a labeling agent capable of detection and which couples with the first composition; and detecting the label with a detection means via the endoscope, laparoscope, or intravascular catheter or during the surgical procedure.
A variation of the above embodiment is when the first composition comprises biotinylated antibody or fragment and the second composition comprises biotin conjugated with fluorescent agent or dye. After the first agent accretes at the targeted lesion and prior to injecting the second composition, the patient is injected with a clearing composition comprising an agent to clear circulating biotinylated antibody or fragment.
In another aspect, the invention provides an improved method for detection of lesions in a patient to undergo an endoscopic, laparoscopic, intravascular catheter or surgical procedure. The method comprises injecting a patient to undergo such a procedure with a first composition comprising an antibody or fragment which preferentially accretes at the lesion and which is labeled with an agent capable of detection with a photoscanning or magnetic resonance imaging device; imaging the lesion with the photoscanning or magnetic resonance device after the labeled antibody or fragment accretes at the lesion; injecting the patient with a second composition comprising an agent capable of detection by an endoscope, laparoscope, intravascular catheter or detection device, such as a hand-held or portable detection means, and which preferentially accretes at the lesion, wherein the agent of the second composition is the same as or different from the agent of the first composition, and allowing the agent to accrete at the lesion; and using the image of the lesion to determine the site for detecting the agent with a close-range detection means provided via the endoscope, laparoscope, or intravascular catheter or during the surgical procedure.
In another aspect, the invention provides an improved method for detection of lesions in a patient to undergo an endoscopic, laparoscopic, intravascular catheter or surgical procedure. The method comprises injecting a patient parenterally with an antibody fragment or subfragment specific to the lesion and which is labeled with a first labeling agent capable of detection using a detection device, and with an indifferent antibody fragment from the same or different species as that used to prepare the specific antibody fragment, the indifferent fragment being labeled with a second labeling agent capable of being independently detected using a detection device, the labeling being so effected that the kinetics and distribution of the labeled specific antibody fragment and the labeled indifferent antibody fragment in the patient are substantially the same during the time period required for detection, wherein at least one of the labeling agents is a photoactive dye; and during the procedure detecting for presence of the labeling agents in the patient with the detection device, the level of activity of the labeled indifferent antibody fragment being used to determine the distribution of background activity due to non-targeted specific antibody fragment, whereby the activity of substantially only the targeted lesion-localized specific antibody fragment is determined and said lesion is thereby detected and localized.
In another aspect, the invention provides an injectable composition, which comprises a substantially monospecific antibody having a specific immunoreactivity of at least 70% to a marker produced by or associated with a lesion and a cross-reactivity to non-lesion-associated antigens of less than 25%; the protein being labeled with a labeling agent capable of detection with a detection device, said labeling being effected to an extent sufficient to reduce the specific immunoreactivity of the antibody by from 5 to 33%; indifferent protein from the same or different species as that used to prepare said specific antibody, said indifferent protein being labeled with a labeling agent capable of independent detection by the detection device; and a pharmaceutically acceptable injection vehicle wherein at least one of the labeling agents is a photoactive dye.
In another aspect, the invention provides an improved method of detection and treatment of lesions. The method comprises injecting a patient with composition comprising a protein conjugated to an agent capable of photoactivated to emit Auger electrons or other ionizing radiation, and, optimally, to an agent capable of detection, wherein the protein conjugate accretes preferentially at the target lesion; and activating the photoactivatable agent and detecting the optional agent capable of detection.
In another aspect, the invention provides an improved method of obtaining biopsy samples, comprising injecting a patient subject parenterally with an effective amount of a labeled antibody fragment or subfragment, which specifically binds an antigen produced by or associated with a lesion, scanning the accessed interior of the patient at close range with a detection means for detecting the presence of the labeled antibody fragment or subfragment, locating the sites of accretion of the labeled antibody fragment or subfragment by detecting elevated levels of the labeled antibody fragment or subfragment at such sites with the detection means, and inserting a biopsy implement into one or more located sites of elevated accretion to obtain a biopsy sample.
In another aspect, the invention provides a method of detection of lesions during an endoscopic, laparoscopic, intravascular catheter, or surgical procedure, comprising injecting a patient who is to undergo such a procedure with a bispecific antibody F(ab)2 or F(abxe2x80x2)2 fragment, wherein the bispecific antibody fragment has a first antibody binding site which specifically binds to an antigen produced or associated with a lesion, such as a tumor- or pathogen-associated antigen, and has a second antibody binding site which specifically binds to a hapten, permitting the antibody fragment to accrete, optionally clearing non-targeted antibody fragments using a galactosylated anti-idiotype clearing agent if the bispecific fragment is not largely cleared from circulation within about 24 hours of injection, injecting hapten, preferably a bivalent hapten, wherein each hapten is a chelate or conjugate of a diagnostic radioisotope such as Tc-99m, I-123, In-11, Ga-67 or the like, or of a MRI image enhancing agent, e.g., Gadolinium ions, Lanthanum ions or the like, or other comparable label, which quickly localizes at the target site and clears through the kidneys, detecting the presence of the hapten by detecting elevated levels of accreted label at the target sites with detection means within 48 hours of the first injection, and conducting the procedure.